Multiple mesodermal lineage differentiation potentials for adipose tissue-derived stromal cells and uses thereof

ABSTRACT

The invention relates to methods and compositions for the differentiation of stromal cells from adipose tissue into hematopoietic supporting stromal cells and myocytes of both the skeletal and smooth muscle type. The cells produced by the methods are useful in providing a source of fully differentiated and functional cells for research, transplantation and development of tissue engineering products for the treatment of human diseases and traumatic tissue injury repair.

CROSS-REFERENCE TO RELATED APPLICATION

This Application claims the benefit of U.S. Provisional Application No.60/149,849 filed on Aug. 19, 1999.

FIELD OF INVENTION

This invention relates to methods and compositions for thedifferentiation of stromal cells from adipose tissue into hematopoieticsupporting stromal cells and myocytes of both the skeletal and smoothmuscle types.

BACKGROUND OF INVENTION

The neonatal period in human development is characterized by thepresence of “stem” cells with the potential to develop along multipledifferentiation pathways. The terminal differentiation of these cells isdetermined by cytokine and hormonal cues which co-ordinate organogenesisand tissue architecture. Murine embryonic stem cells have been isolatedand studied extensively in vitro and in vivo. Using exogenous stimuli invitro, investigators have induced ES cell differentiation along multiplelineage pathways. These include neuronal, B lineage lymphoid, andadipocytes (Dani et al. (1997) J. Cell Sci. 110: 1279; Remoncourt et al.(1998) Mech. Dev. 79:185; O'Shea K S (1999) Anat. Rec. 257:32). The EScells have been manipulated in vivo by homologous recombinationtechniques to generate gene specific null or “knock-out” mice (Johnson RS (1989) Science 245:1234). Once ES cell clones lacking a specific geneare isolated, they are transplanted into a fertilized murine zygote. Theprogeny of this isolated ES cell can develop into any and all murinetissues in a coordinated manner.

A stem cell must meet the following criteria: (1) ability of a clonalstem cell population to self-renew; (2) ability of a clonal stem cellpopulation to generate a new, terminally differentiated cell type invitro; and (3) ability of a clonal stem cell population to replace anabsent terminally differentiated cell population when transplanted intoan animal depleted of its own natural cells.

Multipotential stem cells exist in tissues of the adult organism. Thebest characterized example of a “stem cell” is the hematopoieticprogenitor isolated from the bone marrow and peripheral blood. Seminalstudies by Trentin, Till and McCulloch (McCulloch et al. (1996) Proc.Can. Cancer Conf. 6:356-366; Curry et al. (1967) J. Exp. Med.125:703-720) examined lethally irradiated mice. In the absence oftreatment, these animals died because they failed to replenish theircirculating blood cells; however, transplantation of bone marrow cellsfrom a syngeneic donor animal would rescue the host animal. The donorcells were responsible for reconstituting all of the circulating bloodcells. A wealth of elegant studies have gone on to demonstrate thatdonation of a finite number of undifferentiated hematopoietic stem cellsis capable of regenerating each of the eight or more different bloodcell lineages in a host. This work has provided the basis for bonemarrow transplantation, a widely accepted therapeutic modality for thetreatment of cancer and inborn errors of metabolism in man. Thus,hematopoietic stem cells remain present in the normal human bone marrowthroughout life; they are not limited to the neonatal period.

The recent development of entire organisms from a single donor cell areconsistent with this hypothesis. The “Dolly” experiment showed thatcells isolated from an ovine mammary gland could develop into a maturesheep (Pennisi & Williams (1997) Science 275:1415-1416). In similarmurine studies, cells derived from the corpus luteum of the ovary coulddevelop into a mature mouse (Pennisi (1998) Science 281:495). Thesestudies suggest that stem cells with the ability to differentiate intoany and all cell types continue to exist in the adult organism. Thus,“embryonic” stem cells may be retained throughout life.

In vitro experiments using cell lines of embryonic origin indicate thata mesodermal stem cell may exist. Work by Taylor and colleagues in thelate 1970's demonstrated that murine embryonic fibroblasts such asC3H10T1/2 or 3T3 cells would differentiate along multiple mesodermallineage pathways following exposure to 1 to 10 μM of 5′-azacytadine(Constantinides et al. (1977) Nature 267:364; Jones & Taylor (1980) Cell20:85). Within 2 to 4 weeks, isolated clones displayed a morphologyconsistent with adipocyte, myocyte, chondrocyte or osteoblastdifferentiation. Biochemical data provided additional support for theidentification of each of these lineages. This finding provided thebasis for the identification of the master-regulatory transcriptionfactor for skeletal muscle differentiation, myoD (Lassar (1986) Cell47:649).

The adult bone marrow microenvironment is the potential source for thesehypothetical mesodermal stem cells. Cells isolated from adult marrow arereferred to by a variety of names, including stromal cells, stromal stemcells, mesenchymal stem cells (MSCs), mesenchymal fibroblasts,reticular-endothelial cells, and Westen-Bainton cells (Gimble et al.(1996) Bone 19:421-428). In vitro studies have determined that thesecells can differentiate along multiple mesodermal or mesenchymal lineagepathways. These include, but are not limited to, adipocytes (fat cells)(Gimble et al. (1990) Eur. J. Immunol 20:379-386; Pittenger et al.(1999) Science 284:143-147; Nuttall et al. (1998) JBMR 13:371-382; Parket al. (1999) Bone 24:549-554), chondrocytes (cartilage forming cells)(Dennis et al. (1999) JBMR 14:700-709), hematopoietic supporting cells(Gimble et al. (1990) Eur. J. Immunol. 20:379-386), myocytes (skeletalmuscle) (Phinney (1999) J. Cell. Biochem. 72:570-585), myocytes (smoothmuscle) (Remy-Martin et al. (1999) Exp. Hematol. 27:1782-1795), andosteoblasts (bone forming cells) (Beresford (1989) Clin Orthop Res240:270-280; Owen (1988)J. Cell. Sci. 10:63-76; Dorheim et al. (1993) J.Cell. Physiol. 154:317-328; Haynesworth et al. (1992) Bone 13:81-88,Kuznetsov et al. (1997) JBMR 12:1335-1347). The bone marrow has beenproposed as a source of stromal stem cells for the regeneration of bone,cartilage, muscle, adipose tissue, and other mesenchymal derived organs.The major limitations to the use of these cells are the difficulty andrisk attendant upon bone marrow biopsy procedures and the accompanyingloss of memory B cells and hematopoietic stem cells with presentharvesting procedures.

Another viable alternative to the use of bone marrow multipotential stemcells is stromal cells which can differentiate along multiplemesenchymal lineages. Methods and compositions are needed for theconsistent and quantitative differentiation of adipose derived stromalcells into various cell types including for example hematopoieticstromal cells and skeletal and smooth muscle myocytes.

SUMMARY OF INVENTION

Compositions and methods for the differentiation of adipocytes areprovided. Generally, the present invention provides methods andcompositions for consistent and quantitative induction of stromal cellsderived from subcutaneous, mammary, gonadal, or omental adipose tissuesinto the following fully differentiated and functional mesodermal celllineages: hematopoietic supporting stromal cells, skeletal myocytes, andsmooth muscle myocytes (myofibroblasts).

The compositions include a variety of chemical components which act asmitogens and differentiation inducing agents for the plated stromalcells and yield production of the desired cell type. The mitogens andinducing agents include, but are not limited to, interleukins, flt-3ligand, stem cell factor, macrophage-colony stimulating factor,granulocyte-monocyte colony stimulating factor, erythropoietin,thrombopoietin, osteoprotegerin ligand, dexamethasone, hydrocortisone,1,25 dihydroxy vitamin D₃, 2-mercaptoethanol, glutamine, 5′-azacytadine,amphotericin, transforming growth factor β and fibroblast growth factor.

The invention provides methods for determining the ability of thesecompositions to direct the differentiation and function of theadipose-derived stromal cells, for the transduction of viral vectorscarrying regulatory genes into stromal cells, for the transfection ofplasmid vectors carrying regulatory genes into stromal cells, for thetracking and detection of functional proteins encoded by these genes,and for developing biomechanical carriers for the re-introduction ofthese cells into living organisms.

The invention also provides methods and compositions which have utilityin drug discovery for compounds and proteins with relevance to a widespectrum of disease states including, but not limited to, aplasticanemia, muscular dystrophy, radiation poisoning, neuropathic musculardegeneration, urogenital malformations, and gastrointestinalmalformations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Human adipose-derived stromal cells were cultured in 6 wellplates until confluent and quiescent in DMEM/F 10 (1:1 vol:vol), 10%fetal bovine serum, penicillin 100 units/ml, streptomycin 100 μg/ml, and7.5 mM HEPES pH 7.2. The cells were then incubated in DMEM/F 10 (1:1vol:vol), 2% fetal bovine serum, penicillin 100 units/ml, streptomycin100 μg/ml, and 7.5 mM HEPES pH 7.2 containing 100 ng/mllipopolysaccharide (LPS). Cells were harvested immediately (time “0”) orafter 4 hours (time “4”) for total RNA extraction and isolation using aphenol/chloroform/acid procedure (Chomczynski & Sacchi (1987) AnalyticalBiochem 162:156-159). Equal aliquots of total RNA were reversetranscribed and amplified by polymerase chain reaction with thefollowing primer sets specific for the indicated human mRNAs; the actinprimers served as a positive control for equal loading between samples:

Actin Forward 5′ AGTAACAGCCCACGGTGTTC 3′ Reverse 5′AGCCTCCGAAAGGAAATTGT 3′ Interleukin 6 (IL-6)  Forward 5′GTAGCCGCCCCACACAGACAGCC 3′ Reverse 5′ GCCATCTTTGGAAGGTTCAGG 3′Interleukin 8 (IL-8)  Forward 5′ TCTGCAGCTCTGTGTGAAGGT 3′ Reverse 5′TGAATTCTCAGCCCTCTTCAA 3′ Granulocyte Colony Stimulating Factor (G-CSF)Forward 5′ AGCTTCCTGCTCAAGTGCTTAGAG 3′ Reverse 5′TTCTTCCATCTGCTGCCAGATGGT 3′ Macrophage Colony Stimulating Factor (M-CSF)Forward 5′ TTGGGAGTGGACACCTGCAGTCT 3′ Reverse 5′CCTTGGTGAAGCAGCTCTTCAGCC 3′Granulocyte/Monocyte Colony Stimulating Factor (GM-CSF) Forward 5′GTCTCCTGAACCTGAGTAGAGACA 3′ Reverse 5′ AAGGGGATGACAAGCAGAAAGTCC 3′Flt3 Ligand Forward 5′ TGGAGCCCAACAACCTATCTC 3′ Reverse 5′GGGCTGAAAGGCACATTTGGT 3′ Leukemia Inhibitory Factor (LIF) Forward 5′AACAACCTCATGAACCAGATCAGGAGC 3′ Reverse 5′ATCCTTACCCGAGGTGTCAGGGCCGTAGG 3′

The resulting PCR products were electrophoresed on a 2% agarose gel,stained with ethidium bromide and photographed.

Table 1. Characterization of Adipose Derived Stromal Cell SurfaceMarkers Based on Antibody and PCR Detection. The listed cell surfaceproteins and genes analyzed in human adipose derived stromal cells isbased on immunohistochemical staining, flow cytometry, and/or bypolymerase chain reaction. Markers are divided among those expressed(listed as “positive”) and not expressed (listed as “negative”).Table 2. Cytokines Expressed by Adipose-Derived Stromal CellsConstitutively or Following Endotoxin (LPS) Induction. The listedcytokines were analyzed in total RNA isolated from human adipose derivedstromal cells following induction with 100 ng/ml of lipopolysaccharide.The cytokines listed in the table were detected using theoligonucleotide primers listed. All cytokines were expressed either in aconstitutive or inducible manner.Table 3. Quantitative ELISA (pg/ml) LPS Induction of Adipose-DerivedStromal Cell Secreted Cytokines. The listed cytokines were assayed inconditioned medium from human adipose derived stromal cells induced for0 to 24 hours with 100 ng/ml of LPS. All cytokines were detected byenzyme linked immunoassay (ELISA) and are expressed as pg/ml ofconditioned medium. Those cytokines indicated by an “*” demonstratedsignificant increases relative to the 0 hour time point within the 24hour induction period based on one way analysis of variance.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for thedifferentiation and culture of adipose tissue-derived stromal cells into(a) hematopoietic supporting stromal cells, (b) skeletal myocytes, and(c) smooth muscle myocytes (myofibroblasts).

Adipose tissue offers a potential alternative to the bone marrow as asource of multipotential stromal stem cells. Adipose tissue is readilyaccessible and abundant in many individuals. Obesity is a condition ofepidemic proportions in the United States, where over 50% of adultsexceed the recommended BMI based on their height. Adipocytes can beharvested by liposuction on an outpatient basis. This is a relativelynon-invasive procedure with cosmetic effects which are acceptable to thevast majority of patients. It is well documented that adipocytes are areplenishable cell population. Even after surgical removal byliposuction or other procedures, it is common to see a recurrence ofadipocytes in an individual over time. This suggests that adipose tissuecontains stromal stem cells which are capable of self-renewal.Pathologic evidence suggests that adipose-derived stromal cells arecapable of differentiation along multiple mesenchymal lineages. The mostcommon soft tissue tumor, liposarcomas, develop from adipocyte-likecells. Soft tissue tumors of mixed origin are relatively common. Thesemay include elements of adipose tissue, muscle (smooth or skeletal),cartilage, and/or bone. Just as bone forming cells within the bonemarrow can differentiate into adipocytes or fat cells, theextramedullary adipocytes are capable of forming bone. In patients witha rare condition known as paroxysmal osseous heteroplasia, subcutaneousadipocytes form bone for unknown reasons (Kaplan (1996) Arch. Dermatol.132:815-818).

Adult human extramedullary adipose tissue-derived stromal cellsrepresent a stromal stem cell source which can be harvested routinelywith minimal risk to the patient. They can be expanded ex vivo,differentiated along unique mesodermal lineage pathways, geneticallyengineered, and re-introduced into individuals as either an autologousor allogeneic transplantation. This invention presents examples ofmethods and composition for the isolation, characterization, anddifferentiation of adult human extramedullary adipose tissue stromalcells along the mesodermal lineages and outlines their use for thetreatment of a number of human conditions and diseases.

The cells produced by the methods of invention are useful in providing asource of fully differentiated and functional cells for research,transplantation, and development of tissue engineering products for thetreatment of human disease and traumatic injury repair. Thus, in oneaspect, the invention provides a method for differentiating adiposetissue-derived stromal cells into one of three functionally distinctmesodermal cell lineages: hematopoietic supporting cells, myocytes(skeletal), and myocytes (smooth muscle myofibroblasts) comprising:culturing said cells in a composition which comprises a medium (a)capable of supporting the growth of stromal cells and hematopoieticcells in co-culture with factors present capable of inducing stromalexpression of hematopoietic growth factors or the addition of exogenousgrowth factors directly; (b) capable of supporting the growth anddifferentiation of stromal cells into functional and proliferatingskeletal myocytes; and (c) capable of supporting the growth anddifferentiation of stromal cells into functional and proliferatingsmooth muscle myocytes or myofibroblasts.

In another aspect, the invention provides compositions for thedifferentiation of adipose tissue-derived stromal cells into each of thethree different mesodermal derived lineages. Such compositions comprise:

(a) adipose tissue-derived stromal cells, a medium capable of supportingthe growth of the stromal cells, and growth factors and agents capableof inducing stromal cell expression of hematopoietic growth factors orexogenous hematopoietic growth factors or non-peptide factorsthemselves.

(b) adipose tissue-derived stromal cells, a medium capable of supportingthe growth of the stromal cells, and amounts of 5′ azacytadine and/oramphotericin or other agents sufficient to induce the differentiation ofsaid stromal cells into skeletal muscle myocytes.

(c) adipose tissue-derived stromal cells, a medium capable of supportingthe growth of the stromal cells, and amounts of transforming growthfactor β or other peptide growth factors sufficient to induce thedifferentiation of said stromal cells into smooth muscle myocytes ormyofibroblasts.

The methods comprise incubation of isolated adipose tissue-derivedstromal cells, plated at densities of about 1,000 to 25,000 cells/cm² inmedium consisting of the following for each lineage:

-   -   (a) Hematopoietic supporting stromal cell—Glucose, hematopoietic        inducing cytokines, including but not limited to,        interleukins-1,3,6,7, 11, 12, stem cell factor, flt-3 ligand,        macrophage colony stimulating factor, granulocyte-monocyte        colony stimulating factor, thrombopoietin, erythropoietin,        osteoprotegerin ligand, 1,25 dihydroxy vitamin D₃, and        2-mercaptoethanol. The medium may also contain hydrocortisone,        dexamethasone, and osteoprotegerin ligand. Cells are maintained        at temperatures of 33° C. (for myeloid cells) or 37° C. (for        B-lineage lymphoid cells).    -   (b) Myocytes, Skeletal—Glucose, 5′-azacytadine or amphotericin        for a limited exposure period with manipulation of fetal bovine        serum of concentrations between 0% and 20%. The medium may also        include, but is not limited to, an antibiotic (as for example        penicillin or streptomycin), glutamine, sodium pyruvate, and        2-mercaptoethanol.    -   (c) Myocytes, Smooth Muscle/Myofibroblasts—Glucose and 10% fetal        bovine serum in the presence of a collagen, gelatin, laminin,        fibronectin or other susbtratum or 3-dimensional matrix.

“Adipose stromal cells” refers to stromal cells that originate fromadipose tissue. By “adipose” is meant any fat tissue. The adipose tissuemay be brown or white adipose tissue, derived from subcutaneous,omental/visceral, mammary, gonadal, or other adipose tissue site.Preferably, the adipose is subcutaneous white adipose tissue. Such cellsmay comprise a primary cell culture or an immortalized cell line. Theadipose tissue may be from any organism having fat tissue. Preferably,the adipose tissue is mammalian, most preferably the adipose tissue ishuman. A convenient source of adipose tissue is from liposuctionsurgery, however, the source of adipose tissue or the method ofisolation of adipose tissue is not critical to the invention. If stromalcells are desired for autologous transplantation into a subject, theadipose tissue will be isolated from that subject.

“Hematopoietic supporting stromal cell” refers to stromal cells that arecapable of supporting the proliferation and maturation of hematopoieticprogenitor, also known as hematopoietic stem cells, derived from bonemarrow, spleen, peripheral blood, or umbilical blood, either CD34+ orCD34−. Co-cultures of hematopoietic supporting stromal cells withhematopoietic progenitors would result in the production of bothadherent and non-adherent populations of hematopoietic blood cells,including but not limited to myeloid (macrophage, neutrophil,osteoclast), erythroid (red blood cells), lymphoid (B-lymphoid,T-lymphoid), and platelet (megakaryocyte), as well as eosinophils,basophils, mast cells and other circulating blood cell types. Growth anddifferentiation of hematopoietic cells will be determined by assayswhich include, but are not limited to, those that assess the surfaceexpression of characteristic blood cell lineage specific proteins (suchas CD45 for B lineage lymphocytes, T cell receptor for T lineagelymphocytes, Mac-I/LFA11 for macrophages, tartrate resistant acidphosphatase for osteoclasts). Proliferation of hematopoietic stem cellswill be assessed in vitro by evidence that co-culture derivedhematopoietic cells can continue to expand to multiple blood celllineages when plated onto a fresh hematopoietic supporting stromal celllayer and in vivo based on the ability of co-culture derivedhematopoietic cells to repopulate the bone marrow and rescue a lethallyirradiated animal host lacking its own blood cells.

“Myocytes (skeletal)” refers to cells that are capable of expressingcharacteristic biochemical markers of skeletal muscle, including but notlimited to the transcription factors myoD and myogenin, skeletal actin,myosin light chain kinase, and myosin heavy chain kinase, characteristicmorphologic markers of skeletal muscle, including but not limited tomultinucleated complexes and sarcomeres, and able to exhibit contractilefunction spontaneously or in response to exogenous factors such asacetylcholine.

“Myocytes (smooth muscle, myofibroblasts)” refers to cells that arecapable of expressing characteristic biochemical markers of smoothmuscle, including but not limited to α-smooth muscle actin, fibronectin,and β-1 integrin, characteristic morphologic markers of smooth muscle,including but not limited to the formation of stress fibers in culture,and able to exhibit characteristic smooth muscle functions, includingbut not limited to the generation of tensile stress on collagen latticesin vitro.

“Hematopoietic growth factors” refers to cytokines, hormones and otherprotein agents. These may be derived directly from stromal cells in theco-culture system or added to co-cultures at concentrations determinedby the investigator and obtained as enriched or purified proteinsdeveloped from recombinant or natural sources. These will include butare not limited to the following cytokines and hormones: interleukin 7for the growth of B lineage lymphocytes; stem cell factor for allhematopoietic lineages; M-CSF for macrophages and osteoclasts;osteoprotegerin ligand for osteoclasts; erythropoietin for erythrocytes;thrombopoietin for platelets and megakaryocytes; interleukin 6 forplatelets, megakaryocytes, and B lineage lymphocytes. Optimalconcentrations and length of treatment may be determined by thepractitioner through the use of known assays for the differentiation ofeach blood cell lineage.

“Non-peptide growth factors” refers to steroids, retinoids and otherchemical compounds or agents which induce the differentiation of bloodcell lineages. It is generally recognized that concentrations may vary.Moreover, it is generally recognized that the compounds or agents willbe added in amounts sufficient to stimulate differentiation. Generally,however, these will be used at concentrations ranging from about 1 nM toabout 100 nM for 1,25 dihydroxy vitamin D₃, about 1 nM to about 100 nMdexamethasone, about 1 nM to about 100 nM hydrocortisone, about 1 nM toabout 100 nM retinoic acid, about 1 nM to about 100 nM 9-cis retinoicacid, or at concentrations to be determined and optimized by thepractitioner.

Amounts of 5′ azacytadine and/or amphotericin sufficient to inducedifferentiation refers to concentrations of 5′ azacytadine andamphotericin, that when supplied in a medium capable of supporting thegrowth of stromal cells (e.g. NIH-3T3, C3H 10T1/2, human adiposetissue-derived stromal cells and the like), will induce thedifferentiation of said stromal cells into skeletal muscle myoblasts andmyocytes over a period of about 1 to 6 weeks. Typical use concentrationsfor 5′azacytadine range from about 1 μM to about 30 μM. Typical useconcentrations for amphotericin range from about 10 ng/ml to about 100ng/ml. Optimal concentrations and length of exposure may be determinedby the practitioner through the use of known assays for thedifferentiation of skeletal muscle myoblasts. Such assays include, butare not limited to, those that assess the morphological or biochemicalcharacteristics associated with skeletal muscle (e.g., formation ofmultinucleated myotubules, expression of myosin heavy chain, expressionof myoD at the protein or RNA level).

“Amounts of transforming growth factor β or other peptide growth factorssufficient to induce differentiation” refers to concentrations oftransforming growth factor β or other peptides, that when supplied tomedium capable of supporting the growth of stromal cells (e.g. NIH-3T3,C3H 10T1/2, human adipose tissue-derived stromal cells and the like),will induce the differentiation of said stromal cells into smooth musclemyocytes or myofibroblasts over a period of 1 day to 6 weeks. Typicaluse concentrations for transforming growth factor β range from about 20ng/ml to about 40 ng/ml. Typical use concentrations for fibroblastgrowth factor range from about 20 ng/ml to about 40 ng/ml. Optimalconcentrations and length of exposure may be determined by thepractitioner through the use of known assays for the differentiation ofsmooth muscle myoblasts. Such assays include, but are not limited to,those that assess the morphological or biochemical characteristicsassociated with smooth muscle (e.g., expression of a smooth muscle actinand fibronectin, generation of contractile forces when placed incollagen lattices in the presence of thrombin or lysophosphatidic acid).

Any medium capable of supporting stromal cells in tissue culture may beused. Media formulations that will support the growth of fibroblastsinclude, but are not limited to, Dulbecco's Modified Eagle's Medium(DMEM), alpha modified Minimal Essential Medium (αMEM), and Roswell ParkMemorial Institute Media 1640 (RPMI Media 1640) and the like. Typically,0 to 20% Fetal Bovine Serum (FBS) will be added to the above media inorder to support the growth of stromal cells and hematopoietic cells.However, a defined medium could be used if the necessary growth factors,cytokines, and hormones in FCS for stromal cell and hematopoietic cellgrowth are identified and provided at appropriate concentrations in thegrowth medium.

Media useful in the methods of invention may contain one or morecompounds of interest, including, but not limited to, antibiotics,compounds that are mitogenic for hematopoietic; stem cells,differentiation inducing for hematopoietic stem cells, and/or mitogenicor differentiative for stromal cells. Example of antibiotics useful inthe invention include but are not limited to penicillin andstreptomycin. Penicillin is typically used at about 10 units/ml to about200 units/ml. Streptomycin is typically used at about 10 μg/ml to 200μg/ml. Examples of hematopoietic mitogenic factors include but are notlimited to stem cell factor and interleukin 3; hematopoieticdifferentiation inducing factors include but are not limited to 1,25dihydroxy vitamin D₃, interleukin 7, and osteoprotegerin ligand; stromalcell mitogens include but are not limited to transforming growth factorβ; and stromal cell differentiating factors include but are not limitedto dexamethasone, hydrocortisone, transforming growth factor β, and thelike.

“Adipose tissue-derived stromal cells, a medium capable of supportingthe growth of the stromal cells, and growth factors and agents capableof inducing stromal cell expression of hematopoietic growth factors orexogenous hematopoietic growth factors or non-peptide factorsthemselves” refers to growth factors, both peptide and chemical incomposition, which enhance the proliferation and maturation ofhematopoietic stem cells in vitro and in vivo. These include, but arenot limited to, interleukin 1, interleukin 3, interleukin 6,interleukin. 7, interleukin 11, macrophage colony stimulating factor(M-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF), stemcell factor, flt3 ligand, thrombopoietin, erythropoietin,osteoprotegerin ligand, dexamethasone, hydrocortisone, and 1,25dihydroxy vitamin D₃. The concentrations of these factors and the lengthof time of exposure will be determined and optimized by theinvestigators. Interleukins, M-CSF, GM-CSF, Flt 3 ligand and stem cellfactor are used at about 5 pg/ml to about 1 ng/ml. Thrombopoietin istypically used at concentrations ranging from about 5 pg/ml to about 1ng/ml. Erythropoietin is used at about 5 units/ml to about 1000units/ml. Osteoprotegerin ligand is used at about 5 pg/ml to about 1ng/ml. Dexamethasone, hydrocortisone, and 1,25 dihydroxy vitamin D₃ areat concentrations from about 1 nM to about 100 nM. Optimalconcentrations and treatment times will be determined by monitoring theproduction of specific circulating blood cell lineages in theco-cultures of hematopoietic stem cells and adipose tissue-derivedstromal cells. It is generally recognized that these factors will beadded in amounts sufficient to stimulate differentiation. Such assaysand indices include, but are not limited to, those that assess themorphological or biochemical characteristics of the cells, such as theexpression of cell surface proteins unique to specific blood celllineages by flow cytometry, immunohistochemistry, and/orimmunofluorescent methods, expression of specific mRNAs in the cellpopulation, or by in vivo assessment of the production of hematopoieticstern cells by the co-culture system.

“Adipose tissue-derived stromal cells, a medium capable of supportingthe growth of the stromal cells, and amounts of azacytadine and/oramphotericin or other agents sufficient to induce the differentiation ofsaid stromal cells into skeletal muscle myocytes” refers to thedifferentiation inducing agents used to promote expression of skeletalmuscle specific gene markers and skeletal muscle function in vitro. Themedium comprises fetal bovine serum, antibiotic, L-glutamine, sodiumpyruvate, 2-mercaptoethanol, and 5′ azacytadine or amphotericin. Fetalbovine serum can be used at concentrations ranging from 0.5% to 20%. Theantibiotic typically used, but not limited to, is penicillin orstreptomycin. Penicillin is typically used at concentrations rangingfrom about 10 units/ml to about 200 units/ml. Streptomycin is typicallyused in concentrations ranging from about 10 μg/ml to about 200 μg/ml.L-glutamine and sodium pyruvate are typically used at 0.5 mM to about 2mM. 2-mercaptoethanol is typically used at about 10 μM to about 100 μM.5′ azacytadine is typically used at about 1 μM to about 30 μM.Amphotericin is typically used at about 10 ng/ml to about 100 ng/ml. Theconcentrations of these factors and the length of time of exposure willbe determined and optimized by the investigators. Optimal concentrationand treatment times will be determined by monitoring the morphologic andbiochemical markers characteristic of skeletal muscle. These include,but are not limited to, the production of multi-nucleated myotubules inculture and the expression of muscle specific genes and proteins, suchas muscle transcription factors (myoD, myogenin), myosin light chainkinase, myosin heavy chain kinase, and skeletal muscle actin.

“Adipose tissue-derived stromal cells, a medium capable of supportingthe growth of the stromal cells, and amounts of transforming growthfactor β or other peptide growth factors sufficient to induce thedifferentiation of said stromal cells into smooth muscle myocytes ormyofibroblasts” refers to the differentiation inducing conditions usedto promote the expression of smooth muscle associated gene markers andproteins and smooth muscle function in vitro. The concentrations offactors and the length of time of exposure will be determined andoptimized by the investigators. Optimal concentration and treatmenttimes will be determined by monitoring the morphologic and biochemicalmarkers characteristic of smooth muscle cells. These include, but arenot limited to, the generation of tensile forces by the cells whenplaced in a collagen type I lattice and the expression of smooth musclespecific genes and proteins, such as smooth muscle actin, fibronectin,and laminin.

Preferably, the adipose tissue derived stromal cells are isolated fromthe adipose tissue of the subject into which the final differentiatedcells are to be introduced. However, the stromal cells may also beisolated from any organism of the same or different species as thesubject. Any organism with adipose tissue can be a potential candidate.Preferably, the organism is mammalian, most preferably the organism ishuman.

The adipose tissue derived stromal cells may be stably or transientlytransfected or transduced with a nucleic acid of interest using aplasmid, viral or alternative vector strategy. Nucleic acids of interestinclude, but are not limited to, those encoding gene products which; (1)enhance the growth, differentiation, maturation and proliferation ofhematopoietic cell lineages; examples include osteoprotegerin ligandwhich induces osteoclast development, interleukin 7, which induces Blineage lymphocyte development, erythropoietin, which induceserythrocyte development, and thrombopoietin, which induces plateletdevelopment; (2) enhance the differentiation of skeletal muscle;examples include myoD and myogenin, transcription factors which promotemyotubule formation and expression of skeletal muscle specific genes;(3) enhance the growth, differentiation and maturation of smooth musclecells; examples include transforming growth factor β, which inducessmooth muscle proliferation and extracellular matrix production.

The blood cells produced by in vitro co-cultures of hematopoietic stemcells and adipose tissue derived stromal cells can be introduced aloneor in combination with the stromal component into subjects subject toanemia or limited blood cell production. These may include, but are notlimited to, patients receiving high dose chemotherapy, patientsundergoing bone marrow transplantation, patients suffering from aplasticanemia, patients suffering from sickle cell anemia, and other blooddyscrasias.

Other disorders which may be treated with infusion of stem cellsinclude, but are not limited to, diseases resulting from a failure or adysfunction of normal blood cell production and maturation (i.e.,aplastic anemia and hypoproliferative stem cell disorders); neoplastic,malignant diseases in the hematopoietic organs (e.g., leukemia andlymphomas); broad spectrum malignant solid tumors of non-hematopoieticorigin; autoimmune conditions; and genetic disorders. Such disordersinclude, but are not limited to diseases resulting from a failure ordysfunction of normal blood cell production and maturationhyperproliferative stem cell disorders, including aplastic anemia,pancytopenia, agranulocytosis, thrombocytopenia, red cell aplasia,Blackfan-Diamond syndrome, due to drugs, radiation, or infection,idiopathic; hematopoietic malignancies including acute lymphoblastic(lymphocytic) leukemia, chronic lymphocytic leukemia, acute myelogenousleukemia, chronic myelogenous leukemia, acute malignant myelosclerosis,multiple myeloma, polycythemia vera, agnogenic myelometaplasia,Waldenstrom's macroglobulinemia, Hodgkin's lymphoma, non-Hodgkin'slymphoma; immunosuppression in patients with malignant, solid tumorsincluding malignant melanoma, carcinoma of the stomach, ovariancarcinoma, breast carcinoma, small cell lung carcinoma, retinoblastoma,testicular carcinoma, glioblastoma, rhabdomyosarcoma, neuroblastoma,Ewing's sarcoma, lymphoma; autoimmune diseases including rheumatoidarthritis, diabetes type I, chronic hepatitis, multiple sclerosis,systemic lupus erythematosus; genetic (congenital) disorders includinganemias, familial aplastic, Fanconi's syndrome, Bloom's syndrome, purered cell aplasia (PRCA), dyskeratosis congenita, Blackfan-Diamondsyndrome, congenital dyserythropoietic syndrome I-IV, Chwachmann-Diamondsyndrome, dihydrofolate reductase deficiencies, formamino transferasedeficiency, Lesch-Nyhan syndrome, congenital spherocytosis, congenitalelliptocytosis, congenital stomatocytosis, congenital Rh null disease,paroxysmal nocturnal hemoglobinuria, G6PD (glucose-6-phosphatedehydrogenase) variants 1, 2, 3, pyruvate kinase deficiency, congenitalerythropoietin sensitivity, deficiency, sickle cell disease and trait,thalassemia alpha, beta, gamma, met-hemoglobinemia, congenital disordersof immunity, severe combined immunodeficiency disease (SCID), barelymphocyte syndrome, ionophore-responsive combined immunodeficiency,combined immunodeficiency with a capping abnormality, nucleosidephosphorylase deficiency, granulocyte actin deficiency, infantileagranulocytosis, Gaucher's disease, adenosine deaminase deficiency,Kostmann's syndrome, reticular dysgenesis, congenital leukocytedysfunction syndromes; and others such as osteopetrosis, myelosclerosis,acquired hemolytic anemias, acquired immunodeficiencies, infectiousdisorders causing primary or secondary immunodeficiencies, bacterialinfections (e.g., Brucellosis, Listerosis, tuberculosis, leprosy),parasitic infections (e.g., malaria, Leishmaniasis), fungal infections,disorders involving disproportions in lymphoid cell sets and impairedimmune functions due to aging, phagocyte disorders, Kostmann'sagranulocytosis, chronic granulomatous disease, Chediak-Higachisyndrome, neutrophil actin deficiency, neutrophil membrane GP-180deficiency, metabolic storage diseases, mucopolysaccharidoses,mucolipidoses, miscellaneous disorders involving immune mechanisms,Wiskott-Aldrich Syndrome, alpha 1-antitrypsin deficiency, etc.

The skeletal muscle cells produced by in vitro manipulation of theadipose tissue derived stromal cells can be introduced alone or incombination with a composition matrix to repair muscle defects secondaryto metabolic diseases (muscular dystrophy, myositis), trauma, and disuseatrophy. Such compositions include, but are not limited to, collagenmatrices, poly-lactic polymers, poly-glycolic polymers, alginate, orother solid supports.

The smooth muscle cells produced by in vitro manipulation of the adiposetissue derived stromal cells can be introduced alone or in combinationwith a composition matrix to repair smooth muscle defects. These defectsmay include, but are not limited to, urinary bladder wall abnormalitiesdue to hereditary malformations in neonates or secondary to trauma ortumor invasion in older individuals, gastrointestinal tractabnormalities due to hereditary malformations in neonates or secondaryto trauma or tumor invasion in older individuals, genital tractabnormalities (vaginal) due to hereditary malformations in neonates,secondary to trauma or tumor invasion, or for tissue reconstructivesurgeries in transgender operations, or for the development offunctional large veins for grafting purposes. Composition matrices mayinclude, but are not limited to, collagen matrices such as swineintestinal submucosa, poly-lactic polymers, poly-glycolic polymers,alginate, or other solid supports.

Another object of the invention is to provide methods for theidentification and study of compounds that enhance or inhibit thedifferentiation of adipose tissue derived stromal cells into eitherhematopoietic supporting stromal cells, skeletal muscle myocytes, orsmooth muscle myocytes. Compounds which enhance differentiation. (a)hematopoietic supporting stromal cell function may be of value in thetreatment of blood dyscrasias characterized by decreased production ofcirculating blood cells and improve recovery of patients following highdose chemotherapy; (b) skeletal muscle myocytes may be of value in thetreatment of musculoskeletal diseases secondary to hereditary defects ortrauma; or (c) smooth muscle myocytes may be of value in the treatmentof smooth muscle defects, including those of the urinary bladder(bladder wall), gastrointestinal tract (colon, small intestine), andgenital system (vaginal). Conversely, compounds which inhibitdifferentiation of (a) hematopoietic supporting stromal cells may be ofvalue in the treatment of blood dyscrasias characterized byoverproduction of circulating blood cells, such as polycythemia vera;(b) skeletal muscle may be of value in the treatment of soft tissuetumors of skeletal muscle origin, such as rhabdomyosarcomas; and (c)smooth muscle may be of value in the treatment of soft tissue tumors ofsmooth muscle origin, such as leiomyosarcomas.

Any compound may be tested for its ability to affect the differentiationof adipose tissue derived stromal cells into either hematopoieticsupporting stromal cells, skeletal muscle myocytes, or smooth musclemyocytes. Appropriate vehicles compatible with the compound to be testedare known to those skilled in the art and may be found in the currentedition of Remington's Pharmaceutical Sciences, the contents of whichare incorporated herein by reference.

The features and advantages of the present invention will be moreclearly understood by reference to the following examples, which are notto be construed as limiting the invention.

EXAMPLES Example 1 Expression of Cell Surface Adhesion Molecules andHematopoietic Cytokines by Adipose Tissue-Derived Stromal Cells In Vitro

Stromal cells are isolated from human subcutaneous adipose tissueaccording to methods described in “Methods and Compositions for theDifferentiation of Human Preadipocytes into Adipocytes” Ser. No.09/240,029, filed Jan. 29, 1999. These cells are plated at a density of30,000 cells per cm² in chamber slides, in 6 well tissue culture plates,or in T25 cm² flasks. Cells are maintained in culture for 8 days inDMEM/Ham's F-10 supplemented with 10% fetal bovine serum, penicillin 100units/ml, streptomycin 100 μg/ml, and 7.5 mM HEPES pH 7.2. The surfaceproteins expressed by the stromal cells are determined by immunologictechniques based on immunohistochemistry and/or flow cytometry. Forimmunohistochemical analysis, chamber slides are fixed using 95%ethanol/5% glacial acetic acid and incubated with murine monoclonalantibodies detecting human cell surface proteins. After incubation withan enzyme coupled anti-mouse secondary antibody, evidence of proteinexpression is detected by histochemical reaction. Alternatively, flasksof cells are harvested by trypsin/EDTA digestion and incubated with afluorescent conjugated murine monoclonal antibody detecting a specifichuman surface protein. Cells are examined for fluorescent intensity byflow cytometry. The results of these assays are summarized in Table 1.These studies demonstrate that adipose-derived stromal cells expresscell surface proteins associated with and essential for hematopoieticsupport function by bone marrow stromal cells (Miyake et al. (1990) JExp Med 171:477-488; Miyake et al. (1991) J Exp Med 173:599-607; Miyakeet al. (1991) J Cell Biol 114:557-565, 1991; Jacobsen et al. (1992) JExp Med 176:927-935; Kincade et al. (1993) Curr Top Microbiol Immunol184:215-222; Hayashi et al. (2000) Leuk Lymphoma 38:265-270) theseinclude VCAM1, CD44, integrin β1, integrin α4,5 (VLA-4, VLA-5), and CD9,among others.

The cytokine expression profile of the adipose-derived stromal cells isdetermined following induction with lipopolysaccharide (LPS) orendotoxin, an inflammatory agent capable of inducing hematopoieticcytokines in bone marrow stromal cells (Gimble et al. (1989) Blood74:303-311). Confluent and quiescent cultures of cells are exposed to100 ng/ml LPS for periods of 0 to 24 hours in DMEM medium supplementedwith 2% fetal bovine serum, 100 μg/ml streptomycin, 100 units/mlpenicillin, and 7.5 mM HEPES pH 7.2. The conditioned medium from eachculture is harvested and stored at −80° C. while the total RNA isharvested by the method of Chomczynski and Sacchi (See Anal. Biochem.(1987)162:156-159). The mRNA for the cytokines indicated in Table 2 isdetected by polymerase chain reaction using the oligonucleotide primersets listed below the table. A representative set of reactions isdemonstrated in FIG. 1. The following cytokines demonstrated significantLPS inducible expression of immunoreactive protein based on enzymelinked immunoassay: macrophage colony stimulating factor (M-CSF),granulocyte/monocyte colony stimulating factor (GM-CSF), interleukin 6,7, and 8 (IL-6, 7, 8). The profile of cytokines expressed by the adiposederived stromal cells is consistent that of bone marrow-derived stromalcells capable of supporting myeloid, lymphoid, and osteoclastproliferation and differentiation in vitro (Pietrangeli et al. (1988)Eur. J. Immunol. 18:863-872; Gimble et al. (1989) Blood 74:303-311;Gimble et al. (1992) J. Cell Biochem 50:73-82; Kelly et al. (1998)Endocrinol. 139:2092-2101).

Example 2 Establishment of Myelopoietic Co-Cultures with an AdiposeTissue-Derived Stromal Cells Layer In Vitro

Stromal cells are isolated from human subcutaneous adipose tissueaccording to methods described in “Methods and Compositions for theDifferentiation of Human Preadipocytes into Adipocytes” Ser. No.09/240,029, filed Jan. 29, 1999. These cells are plated at a density of500 to 20,000 cells per cm². Stromal cells are established in thecultures for 1 to 3 days prior to the introduction of hematopoieticprogenitor cells into the co-culture system. Hematopoietic progenitorcells are isolated from one of the following human tissues: bone marrow,umbilical vein/placental blood, peripheral blood, spleen. Alternatively,murine tissues are used. Murine bone marrow cells are harvested byflushing the marrow cavity of 6 to 10 week old mice with DMEM/10% FCSunder sterile conditions. Murine spleen cells are harvested by physicalpassage through a fine metal screen under sterile conditions. One ofthree methods are used to deplete the mixed hematopoietic cellpopulation of its stromal component. As a first alternative, thehematopoietic stem cell population from the blood sample will beenriched by magnetic immunobead purification using anti-CD34 antigenaccording to established techniques. As a second alternative,hematopoietic cells will be enriched by passage of the bone marrow orother blood sample over a sterile G-10 Sephadex or nylon wool column;hematopoietic progenitor cells are eluted while stromal cells areretained. As a third alternative, hematopoietic cells will be enrichedby flow cytometric sorting based on surface protein characteristics. Thehematopoietic cells are washed once and the number of nucleated cells iscounted using a hematocytometer after erythrocyte lysis with 0.3% aceticacid and trypan blue staining. Hematopoietic cells are introduced intothe liquid co-cultures at a ratio of preferably 10 to 100 nucleatedhematopoietic cells per stromal cell plated, more preferably 20 to 30nucleated hematopoietic cells per stromal cell plated, most preferably25 to 30 nucleated hematopoietic cells per stromal cell plated. Cellsare cultured in a medium consisting of DMEM (high glucose), 10% fetalbovine serum supplemented with 10 to 100 nM hydrocortisone or 10% horseserum, 10 to 200 units/ml of penicillin, 10 to 200 μg of streptomycin at33° C. in 5% CO₂. One half of the medium in the co-cultures is replacedevery 3 to 4 days. The number of non-adherent cells in the medium isdetermined by hematocytometer count and/or by flow cytometery. Thesurface antigen characteristics of the non-adherent cells are documentedusing routine antibody markers for the major hematopoietic celllineages. These will include, but are not limited to, Mac-1, Thy 1, IgHeavy chain, Ter-81 (erythroid marker). The number and character of thenon-adherent cell population is determined over a period of up to 10weeks. At the conclusion of the study, the cellular composition of theadherent cell layer is determined by flow cytometric orimmunohistochemical methods. As an alternative approach, thehematopoietic studies are conducted in semi-solid cultures. Cells areprepared as described above, but the hematopoietic progenitor cells areplated in the additional presence of 2.1% methylcellulose. Colonyformation is assessed after a 7 to 14 day period for the presence ofgranulocytes, erythrocytes, macrophages, and monocytes using histologic,morphologic and immunologic criteria.

Example 3 Ability of Adipose Tissue Derived Stromal Cell/HematopoieticProgenitor Cell Co-Cultures to Maintain Proliferation of HematopoieticProgenitors In Vitro

Adipose tissue derived stromal cell/hematopoietic progenitor co-culturesestablished under liquid culture conditions described in Example 1 areused to assess the ability of this system to maintain the proliferationof the hematopoietic progenitor cells in vitro. Co-cultures areestablished using human adipose tissue derived stromal cells and murinehematopoietic progenitors. Cultured cells are transduced with a viralvector expressing a traceable protein marker such as green fluorescentprotein or beta-galactosidase. Alternatively, co-culture cells areidentified by expression of a unique antigen or genetic marker due totheir origin; e.g., the expression of human proteins for the stromalcells, and the expression of a transgenic or male gender specific markerfor the murine hematopoietic cells. Established co-cultures areharvested by limited incubation with trypsin/EDTA and infused into alethally irradiated immunodeficient mouse. Animals will be followed overtime. After 9 to 14 days, mice are sacrificed and their spleens examinedfor the appearance of hematopoietic cell islands or splenic colonyforming units (CFU-S). Alternatively, mice will be maintained for 14days or longer and their circulating blood cell count determined byhematologic and flow cytometric assays. The presence of specific markersof the stromal cells and the donor hematopoietic cells is detected withantibody reagents or specific DNA markers on fixed cells, either by flowcytometry or conventional pathologic/histologic methods. The ability ofthe co-cultured cells to establish CFU-S in the recipient and/or tomaintain the proliferation and maturation of donor blood cells in thehost after 14 days is evidence of the continued expansion of somehematopoietic progenitors in vitro by the adipose tissue-derived stromalcells.

Example 4 Establishment of Lymphopoietic Co-Cultures with an AdiposeTissue-Derived Stromal Cells Layer In Vitro

Stromal cells are isolated from human subcutaneous adipose tissueaccording to methods described in “Methods and Compositions for theDifferentiation of Human Preadipocytes into Adipocytes” Ser. No.09/240,029, filed Jan. 29, 1999. These cells are plated at a density of500 to 20,000 cells per cm². Stromal cells are established in thecultures for 1 to 3 days prior to the introduction of hematopoieticprogenitor cells into the co-culture system. Hematopoietic progenitorcells are isolated from one of the following human tissues: bone marrow,umbilical vein/placental blood, peripheral blood, spleen. Alternatively,murine tissues are used. Murine bone marrow cells are harvested byflushing the marrow cavity of 6 to 10 week old mice with RPMI/10% FCSunder sterile conditions. Murine spleen cells are harvested by physicalpassage through a fine metal screen under sterile conditions. One ofthree methods are used to deplete the mixed hematopoietic cellpopulation of its stromal component, As a first alternative, thehematopoietic stem cell population from the blood sample will beenriched by magnetic immunobead purification using anti-CD34 antigenaccording to established techniques. As a second alternative,hematopoietic cells will be enriched by passage of the bone marrow orother blood sample over a sterile G-10 Sephadex or nylon wool column;hematopoietic progenitor cells are eluted while stromal. cells areretained. As a third alternative, hematopoietic cells will be enrichedby flow cytometric sorting. The hematopoietic cells are washed once andthe number of nucleated cells is counted using a hematocytometer aftererythrocyte lysis with 0.3% acetic acid and trypan blue staining.Hematopoietic cells are introduced into the liquid co-cultures at aratio of preferably 10 to 100 nucleated hematopoietic cells per stromalcell plated, more preferably 20 to 30 nucleated hematopoietic cells perstromal cell plated, most preferably 25 to 30 nucleated hematopoieticcells per stromal cell plated. Cells are cultured in a medium consistingof RPMI1640, prescreened 10% fetal bovine serum, 10 to 200 units/ml ofpenicillin, 10 to 200 μg/ml of streptomycin, 0.5 to 2 mM L-glutamine, 10to 100 μM 2-mercaptoethanol at 37° C. in 5% CO₂. One half of the mediumin the co-cultures is replaced every 3 to 4 days. The number ofnon-adherent cells in the medium is determined by hematocytometer countand/or by flow cytometery. The surface antigen characteristics of thenon-adherent cells are documented using routine antibody markers for themajor hematopoietic cell lineages. These will include, but are notlimited to, Mac-1, Thy 1, Ig Heavy chain, Ter-81 (erythroid marker). Thenumber and character of the non-adherent cell population is determinedover a period of up to 10 weeks. At the conclusion of the study, thecellular composition of the adherent cell layer is determined by flowcytometric or immunohistochemical methods. As an alternative approach,the hematopoietic studies are conducted in semi-solid cultures. Cellsare prepared as described above but the hematopoietic progenitor cellsare plated in the additional presence of 2.1% methylcellulose. Colonyformation is assessed after a 7 to 14 day period for the presence of Blineage lymphoid cells as well as granulocytes, erythrocytes,macrophages, and monocytes using histologic, morphologic and immunologiccriteria. Alternatively, co-cultures and/or semi-solid cultures areestablished as described above with the addition of interleukin 7 atconcentrations to be determined by the practitioner which enhance theproliferation and maturation of B lineage lymphocytes. The techniquesoutlined above are used to assess the affect of this growth factor onthe hematopoietic support function of the adipose tissue derived stromalcell.

Example 5 Establishment of Osteoclastogenic Co-Cultures with an AdiposeTissue-Derived Stromal Cells Layer In Vitro

Stromal cells are isolated from human subcutaneous adipose tissueaccording to methods described in “Methods and Compositions for theDifferentiation of Human Preadipocytes into Adipocytes” Ser. No.09/240,029, Filed Jan. 29, 1999. These cells are plated at a density of500 to 20,000 cells per cm² in 24 well plates. Cells are cultured in amedium consisting of DMEM (high glucose), prescreened 10% fetal bovineserum, 10 to 200 units/ml of penicillin, 10 to 200 μg of streptomycin,0.5 to 2 mM L-glutamine, 0.5 to 2 mM sodium pyruvate, 10 to 100 μM2-mercaptoethanol at 37° C. in 5% CO₂. Three days after the stromalcultures are established, the medium is supplemented with either 10 to100 nM 1,25 dihydroxy vitamin D₃ and or osteoprotegerin ligand atconcentrations determined by the practitioner. Stromal cells areestablished in the cultures for 6 days prior to the introduction ofhematopoietic progenitor cells into the co-culture system.

Hematopoietic progenitor cells are isolated from one of the followinghuman tissues: bone marrow, umbilical vein/placental blood, peripheralblood, spleen. Alternatively, murine tissues are used. Murine bonemarrow cells are harvested by flushing the marrow cavity of 6 to 10 weekold mice with DMEM (high glucose)/10% FCS under sterile conditions.Murine spleen cells are harvested by physical passage through a finemetal screen under sterile conditions. One of three methods are used todeplete the mixed hematopoietic cell population of its stromalcomponent. As a first alternative, the hematopoietic stem cellpopulation from the blood sample will be enriched by magnetic immunobeadpurification using anti-CD34 antigen according to establishedtechniques. As a second alternative, hematopoietic cells will beenriched by passage of the bone marrow or other blood sample over asterile G-10 Sephadex or nylon wool column; hematopoietic progenitorcells are eluted while stromal cells are retained. As a thirdalternative, hematopoietic cells will be enriched by flow cytometricsorting based on surface protein characteristics. The hematopoieticcells are washed once and the number of nucleated cells is counted usinga hematocytometer after erythrocyte lysis with 0.3% acetic acid andtrypan blue staining. Hematopoietic cells are introduced into the liquidco-cultures at a ratio of preferably 10 to 100 nucleated hematopoieticcells per stromal cell plated, more preferably 20 to 30 nucleatedhematopoietic cells per stromal cell plated, most preferably 25 to 30nucleated hematopoietic cells per stromal cell plated. One half of themedium in the co-cultures is replaced every 3 to 4 days. Co-cultures aremaintained in the presence of 1,25 dihydroxy vitamin D₃ orosteoprotegerin ligand continuously after the introduction ofhematopoietic cells.

After a period of co-culture of 6 to 9 days, co-cultures are fixed with0.5 ml 3.7% (vol:vol) formaldehyde in phosphate buffered saline for 5minutes, dried for 30 seconds with acetone:ethanol (50:50, vol/vol), andstained for 10 minutes with 10 mM sodium tartrate, 40 mM sodium acetate(pH 5.0), 0.1 mg/ml naphthol AS-MS phosphate (Sigma N-5000), and 0.6mg/ml fast red violet LB salt (Sigma F-3381). Stained cultures arerinsed in distilled water and stored under 50% glycerol/PBS. The numberof tartrate resistant acid phosphatase positive cells per well isassessed under light microscopy based on the red staining of thecytoplasm. TRAP+ cells are, numerically counted and those with 1-2nuclei are distinguished from those multinucleated cells with ≧3 nucleiper cell. This assay demonstrates the ability of adipose tissue derivedstromal cells to support the differentiation and proliferation ofosteoclastogenic precursors in vitro. This culture procedure is able toexpand and promote differentiation of osteoclasts. This has potentialapplication to rare clinical conditions such as osteopetrosischaracterized by brittle bones where patients fail to produce nativeosteoclasts. This In vitro method offers a means to expand anindividual's own osteoclast progenitors and to promote theirdifferentiation ex vivo. This cell population can be re-infused into theaffected individual with potential short-term or long-term benefit. Thenon-invasive nature of the methodology and the potential to relyexclusively on autologous cells indicates that this procedure could beused repetitively in the treatment of an individual patient.

Example 6 Use of Adipose Tissue Derived Stromal Cell Supported Ex vivoHematopoiesis as a Therapeutic Modality for Bone Marrow Transplant andHematologically Compromised Patients

The co-culture models outlined in Examples 1-4 have the potential to beused to facilitate the recovery of bone marrow function in patientsreceiving high dose chemotherapy, high dose radiation treatment or anyother therapeutic modality which compromises blood cell production andbone marrow function. In advance of any elective procedure, anindividual can donate his or her own adipose tissue and blood cells forsubsequent autologous transplantation. Prior to immunocompromisingprocedures, the individual's own blood cells and stromal co-cultures canbe established and expanded. Following any immunocompromising procedure,the patients own blood cell/stromal cell co-culture can be re-infusedinto the patient according to standard transfusion methodologies. Thiscan be done in the absence or presence of exogenous hematopoieticcytokines, either added to the co-cultures or given directly to thepatient. This approach may accelerate the rate of blood cell productionin the patient, reduce the need for cytokine therapies, and reduce theoverall costs and risk of the chemotherapy or other immunocompromisingprocedure. With the, ex vivo nature of the procedure, it is possible tomanipulate the stromal cells to enhance production of specific bloodcell lineages. Stromal cells transiently expressing interleukin 7, forexample, would facilitate the rapid expansion of B lineage lymphoidcells while stromal cells expressing erythropoietin would favorexpansion of erythrocytes.

As outlined above, the approach is designed primarily for the treatmentof nosocomial induced blood cell dyscrasias. However, large scale exvivo production of autologous stromal/hematopoietic co-cultures is ofpotential value for elective and non-elective surgical proceduresrequiring transfusion intra-operatively and post-operatively. Atpresent, the majority of patients requiring blood transfusion receiveblood products donated by others. This presents risk to the recipient oftransmission of unrecognized infectious disease from the donor. With theability to develop blood cell production ex vivo, an individual canexpand his/her hematopoietic cell population at a capacity which is nolonger limited by the bone marrow cavity volume. Using cell factorytissue culture approaches with recirculating systems, continuousproduction of blood cells by an adipose tissue derived stromalcell/hematopoietic cell co-culture is feasible. This approach has theadvantage of avoiding risks inherent in transfusion of blood from adonor to a recipient; these include the transmission of infectiousdiseases such as HIV, hepatitis, cytomegalovirus, Jacob/Creukzfelddisease, among others.

Example 7 Differentiation of Adipose Tissue Derived Stromal Cells intoSkeletal Muscle Myoblasts

Stromal cells are isolated from human subcutaneous adipose tissueaccording to methods described in “Methods and Compositions for theDifferentiation of Human Preadipocytes into Adipocytes” Ser. No.09/240,029, filed Jan. 29, 1999. These cells are plated at a density of500 to 20,000 cells per cm² in 24 well plates. Cells are cultured in amedium consisting of DMEM (high glucose), prescreened 10% fetal bovineserum, 10 to 200 units/ml of penicillin, 10 to 200 μg of streptomycin,0.5 to 2 mM L-glutamine, 0.5 to 2 mM sodium pyruvate, 10 to 100 μM2-mercaptoethanol at 37° C. in 5% CO₂. Cells are exposed to 1 to 30 μM5′ azacytadine or 10 to 100 ng/ml of amphotericin for periods of 1 to 6days to assure that cells throughout S phase are continuously exposed tothese agents. Following this period, cultures are maintained in theculture medium without azacytadine or amphotericin supplements. Culturesare either continued at the established cell density or sub-cloned bylimit dilution methods to select for cell clones capable of expressingcharacteristic markers of skeletal muscle myoblasts. These cells areselected based on morphologic criteria, specifically, the ability toform multinucleated myotubules in culture; biochemical criteria,specifically, the expression of myosin heavy and light chain kinase,skeletal muscle actin and myosin and the expression of myogenictranscription factors, myoD and/or myogenin.

Example 8 Application of Skeletal Muscle Myoblasts Differentiated fromAdipose Tissue-Derived Stromal Cell

The cells skeletal muscle myoblasts developed in Example 6 can be usedfor tissue engineering purposes in the treatment of myodystrophies,muscle atrophy, and physical loss of skeletal muscle secondary tosurgical procedures for the treatment of cancer or trauma, The ex vivodevelopment of a proliferating population of myoblasts from adiposetissue can be used to supplement and repair skeletal muscle mass inafflicted individuals. Myoblasts can be cultured in biodegradablematrices composed of poly-lactic, poly-glycolic, collagen or othermaterials to form muscle strands. These can then be implanted to anafflicted site and tethered by suture to existing muscle, tendon orbone. Alternatively, ex vivo expanded myoblasts can be geneticallyengineered by viral transduction, plasmid transfection, or other meansto express novel genes. These cells can be injected directly intoexisting muscle sites where these novel gene products will now beexpressed. This approach has direct application to muscular dystrophy,where patients suffer secondary to a mutation in an important skeletalmuscle gene. Likewise, the engineered stromal cells can convert themuscle into a production site for a deficient circulating protein. Forexample, adipose tissue derived stromal cells expressing lipoproteinlipase can be used to treat the many patients with mutations in theirnative lipoprotein lipase gene who are at increased risk of severecardiovascular disease.

Example 9 Differentiation and Expansion of Smooth Muscle Myoblasts fromAdipose Tissue Derived Stromal Cells Ex Vivo

Stromal cells are isolated from human subcutaneous adipose tissueaccording to methods described in “Methods and Compositions for theDifferentiation of Human Preadipocytes into Adipocytes” Ser. No.09/240,029, filed Jan. 29, 1999. These cells are plated at a density of500 to 20,000 cells per cm² in 24 well plates. Cells are cultured in amedium consisting of DMEM (high glucose), prescreened 10% fetal bovineserum, 10 to 200 units/ml of penicillin, 10 to 200 μg of streptomycin,0.5 to 2 mM L-glutamine, 0.5 to 2 mM sodium pyruvate, at 37° C. in 5%CO₂. Cultures are supplemented with transforming growth factor β and/orfibroblast growth factor at concentrations determined by thepractitioner but not to exceed 40 ng/. Cells are maintained in cultureas a monolayer or in a 3-dimensional lattice composed of collagen type Ior other biodegradable material (alginate, synthetic polymer or other).Cells are characterized based on morphologic, biochemical, andfunctional criteria for smooth muscle myoblast differentiation; theseinclude, but are not limited to, expression of smooth muscle actin,fibronectin, laminin, and other extracellular matrix proteins, theability to generate a tensile force as measured by a pressuretransducer, and to organize along a line of force in a 3 dimensionallattice.

Example 10 Application of Smooth Muscle Myoblasts Differentiated fromAdipose Tissue-Derived Stromal Cells Ex Vivo

The smooth muscle myoblasts described under Example 8 can be used totreat conditions where smooth muscle function is compromised. Forexample, over 1000 neonates each year suffer from bladder wallabnormalities. The severity of this disorder is variable but it can bedevastating and requires expensive surgical procedures to accomplish anacceptable repair and an approach to normal function. In many cases, thebladder size is too small or the bladder wall is incompletely formed,necessitating the implantation of prosthetic materials as a bladder wallreplacement. Methods under investigation include the use of swineintestinal submucosa as a replacement material for the bladder wall. Oneissue is whether or not the surgically implanted bladder wall willachieve the appropriate physical and mechanical characteristicsassociated with stretching and retraction. Much of this is mediated byfunctional smooth muscle cells. Current methods implant the SIS materialwithout preimplantation of any smooth muscle cells ex vivo. Surgeonsrely on the recruitment of fibroblasts and myofibroblasts from adjacenttissues, including the omental adipose tissue. With the availablity ofadipose tissue derived stromal cells capable of smooth muscle myoblastdifferentiation, it is possible to pre-incubate SIS material with thesecells ex vivo. The introduction of these cells prior to the surgicalrepair of the bladder wall is expected to accelerate and improve theattainment of appropriate bladder tone. This approach has broadapplication to all elastic soft tissue organs which rely on smoothmuscle cells. These include, but are not limited to, the smallintestine, large intestine, vagina, urethra, and venous blood vessels.The adipose tissue derived stromal cells have potential application tothe surgical repair of defects in any of these organs.

All publications mentioned in the specification are indicative of thelevel of those skilled in the art to which this invention pertains.Publications are herein incorporated by reference to the same extent asif each individual publication was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A medium for differentiating adipose tissue derived stromal cellsinto hematopoietic supporting stromal cells that will proliferate anddifferentiate along the myeloid lineage pathway or the B-lineagelymphoid pathway, said medium comprising: a chemically defined mediumhaving or supplemented with the following components present insufficient amounts to stimulate differentiation (i) 0% to 20% fetalbovine serum (ii) antibiotic (iii) interleukins (iv) stem cell factor(v) flt-3 ligand (vi) macrophage-colony stimulating factor (vii)granulocyte-monocyte colony stimulating factor (viii) erythropoietin(ix) thrombopoietin (x) osteoprotegerin ligand (xi) dexamethasone (xii)hydrocortisone (xiii) 1,25 dihydroxy vitamin D₃ and (xiv)2-mercaptoethanol. 2-21. (canceled)
 22. A medium for differentiatingadipose tissue derived stromal cells into skeletal muscle myocytes, saidmedium comprising: a chemically defined medium having or supplementedwith the following components present in sufficient amounts to stimulatedifferentiation (i) 0.5% to 20% fetal bovine serum (ii) an antibiotic(iii) glutamine (iv) sodium pyruvate (v) 2-mercaptoethanol and (vi) 5′azacytadine or amphotericin. 23-33. (canceled)
 34. A medium fordifferentiating adipose tissue derived stromal cells into smooth musclemyoblasts, said medium comprising: a chemically defined medium having orsupplemented with the following components present in amounts sufficientto stimulate differentiation (i) 0% to 10% fetal bovine serum (ii) anantibiotic (iii) glutamine (iv) sodium pyruvate (v) transforming growthfactor β or fibroblast growth factor and (vi) a 3-dimensional matrixcomposed of a biodegradable material. 35-43. (canceled)
 44. A method fordifferentiating adipose tissue derived stromal cells into hematopoieticsupporting stromal cells which will proliferate and differentiate alongthe myeloid lineage pathway or the B-lineage lymphoid pathway,comprising: a) plating said stromal cells at a density of about 30,000cells per cm² in chamber slides; b) maintaining cells in culture forabout 8 days in a medium containing Dulbecco's Modified Eagle's Medium(DMEM) or Ham's F-10; c) supplementing said medium with: (i) 1 to 20%fetal bovine serum (ii) an antibiotic (iii) interleukins (iv) stem cellfactor (v) flt-3 ligand (vi) macrophage-colony stimulating factor (vii)granulocyte-monocyte colony stimulating factor (viii) erythropoetin (ix)thrombopoietin (x) osteoprotegerin ligand (xi) dexamethasone (xii)hydrocortisone (xiii) 1,25 dihydroxy vitamin D₃ and (xiiii)2-mercaptoethanol; and d) examining the expression of cell surfaceproteins which are consistent with cells of the myeloid lineage orB-lymphoid lineage using a variety of techniques which include but arenot limited to immunohistochemistry, flow cytometry, immunofluorescenceand mRNA expression in cell populations.
 45. The medium of claim 44,wherein said antibiotic is penicillin.
 46. The medium of claim 44,wherein said antibiotic is streptomycin. 47-61. (canceled)
 62. A methodfor differentiating adipose tissue derived stromal cells into skeletalmuscle myocyte cells, comprising: a) plating said stromal cells at adensity of about 500 to about 20,000 cells per cm² in chamber slides; b)maintaining cells in culture for about 8 days in a medium containingDulbecco's Modified Eagle's Medium (DMEM) or Ham's F-10; c)supplementing said medium with: (i) 1 to 10% fetal bovine serum (ii) anantibiotic (iii) glutamine (iv) sodium pyruvate (v) 2-mercaptoethanol;d) exposing cells to azacytadine or amphotericin for 1 to 6 days; e)examining said cells for biochemical phenotypes or markerscharacteristic of skeletal muscle myoblasts.
 63. The method according toclaim 62, wherein said antibiotic is penicillin.
 64. The methodaccording to claim 62, wherein said antibiotic is streptomycin. 65-70.(canceled)
 71. A method for differentiating adipose tissue derivedstromal cells into smooth muscle myoblast cells, comprising: a) platingsaid stromal cells at a density of about 500 to about 20,000 cells percm² in chamber slides; b) maintaining cells in a medium containingDulbecco's Modified Eagle's Medium (DMEM) or Ham's F-10; c)supplementing said medium with: (i) 1 to 10% fetal bovine serum (ii) anantibiotic (iii) glutamine (iv) sodium pyruvate (v) transforming growthfactor β and/or fibroblast growth factor; d) maintaining cells as amonolayer or in a 3-dimensional lattice comprising collagen type I, oralginate or other biodegradable material; and e) characterizing cellsfor biochemical or functional criteria to establish smooth muscledifferentiation.
 72. The method according to claim 71, wherein saidantibiotic is penicillin.
 73. The method according to claim 71, whereinsaid antibiotic is streptomycin. 74-78. (canceled)
 79. A hematopoieticcell made by the method of claim 44.